Agroforest Syst

https://doi.org/10.1007/s10457-019-00408-1 (0123456789().,-volV)
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89().,-volV)

In vitro screening of Algerian steppe browse plants

for digestibility, rumen fermentation profile and methane
mitigation
Lyas Bouazza . Souhil Boufennara . Mustapha Bensaada .
Azzeddine Zeraib . Khalid Rahal . Cristina Saro . Marı́a José Ranilla .
Secundino López

Received: 11 June 2018 / Accepted: 5 June 2019

Ó Springer Nature B.V. 2019

Abstract The aim of this study was to screen the
nutritive value and the effects of anti-nutritional
secondary compounds (condensed tannins) on
in vitro rumen fermentation and methane mitigation
of Algerian steppe browse species: Albizia julibrissin
(pods), Acacia nilotica (pods), Punica granatum
(leaves and pericarp), Vicia faba (leaves), Artemisia
herba-alba (aerial part), Attriplex halimus (leaves) and
Calligonum azel (bark). Chemical composition, and
in vitro digestibility, and rumen fermentation kinetics
and end-products accumulation in batch cultures were
determined. Polyethylene glycol (PEG), a tannin
binding agent was used to measure the biological
activity of tannins. Protein content was high for
A. julibrissin and V. faba and low for the pericarp of
P. granatum and bark of C. azel. The highest concen-
trations of total extractable phenols and tannins were
observed in P. granatum, whereas A. halimus showed
the lowest concentrations. A. nilotica, C. azel and A.
julibrissin showed the highest and A. halimus and A.
L. Bouazza (&)  S. Boufennara  M. Bensaada 
A. Zeraib  K. Rahal
Département de biologie moléculaire et cellulaire, Faculté
des sciences de la nature et de la vie, Université Abbès
Laghrour, 40000 Khenchela, Algeria
e-mail: ilbouazza@yahoo.fr
C. Saro  M. J. Ranilla  S. López
Instituto de Ganaderı́a de Montaña (CSIC-Universidad de
León), Departamento de Producción Animal, Universidad
de León, 24007 León, Spain
herba-alba the lowest total condensed tannin contents.
Vicia faba was the most digestible forage. All the
browse species used in the current study, with the
exception of C. azel bark, can be used as alternative
feedstuffs for ruminant nutrition. The most promising
forage in terms of reduced methane emissions is
Atriplex halimus foliage, because the decreased
methane production is not associated to a reduced
rumen degradation and fermentation of this forage in
the rumen. However, in vivo studies are warranted to
confirm its potential to be included in ruminant diets.
Keywords Nutritive value  Rumen  Roughage 
Tannin  In vitro fermentation  Methane
Introduction
Algerian steppe (30 Mha of land) constitutes a tran-
sition area between the Sahara Desert and the green
belt in the North of the country. The steppe is used
mainly for sheep production, farming local breeds well
adapted to the extreme environmental conditions and
with a particular productive performance. Droughts
occur frequently and have a critical influence on
vegetation, and thus on rangelands. Currently, steppe
rangelands are in a process of degradation due to the
fragility of the physical environment, intensified by
changes in the pastoral methods (Aidoud 1994).

123

Perennial acacia and shrubby plants from Saharan
areas can be used to mitigate desertification, enhanc-
ing soil fixation, restoration of the vegetation and
rehabilitation of rangelands. Trees, especially those of
the Acacia genus, are adapted to arid environments.
Acacia leaves and pods are a potential source of
protein for herbivores, especially when herbaceous
vegetation becomes withered during droughts (Osuji
and Odenyo 1997). However, their content in anti-
nutritional factors (secondary compounds), such as
condensed tannins, limits their use as forage (Waghorn
et al. 2002). Methane emission from ruminants
contributes to the greenhouse gas effect and global
warming (UNFCCC 2005). The digestion of feed by
rumen microbes (archaea, bacteria, protozoa and
fungi) under anaerobic conditions results in the
production of volatile fatty acids (VFA), ammonia,
CO2 and methane (Martin et al. 2010). Rumen
methane represents a loss of 2–12% of the feed energy
(Boadi et al. 2004). Thus, reducing ruminal CH4 will
improve the efficiency of nutrient utilization and
contribute to protect the environment. To mitigate
enteric CH4 emissions from ruminants several dietary
strategies have been suggested (Beauchemin et al.
2008; Patra et al. 2017). Condensed tannins affect
rumen fermentation, and tannin-rich plants can be
used to reduce methane production by ruminants
(Jayanegara et al. 2009; Goel and Makkar 2012;
Naumann et al. 2018). The addition of polyethylene
glycol (PEG) has been used to block the tannin activity
in plants (Getachew et al. 2000a). The objectives of
this study were to screen the nutritive value of
Algerian-steppe browse plants, and to assess the
effects of their tannins on in vitro rumen fermentation
and methanogenesis.
Materials and methods
Plant species
Substrates used in this study were deseeded pods from
Acacia nilotica and Albizia julibrissin, Punica grana-
tum fruits and leaves, aerial part of Artemisia herba-
alba, and leaves from Atriplex halimus and Vicia faba,
all collected from the steppe area of Djelfa province
(34°400 N 3°150 E). Additionally, bark of Calligonum
azel was collected from the Saharan area of Nakhla (El
Oued province, 33°160 N 6°570 E). Samples from
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different specimens were pooled and oven-dried at
55 °C.
Chemical analysis
Dry matter (DM), ash and crude protein (CP) contents
were determined by the methods 934.01, 942.05 and
990.03 of AOAC (2002), respectively. Neutral and acid
detergent fibre and acid detergent lignin were determined
using the ANKOM fibre analyzer following the proce-
dures described by Van Soest et al. (1991). Extraction of
phenolic compounds and colorimetric analyses of phe-
nolics and tannins were performed according to the
procedures described by Makkar (2003).
Animals and collection of rumen fluid
Three cannulated mature Merino sheep
(49.4 ± 4.23 kg body weight) fed with lucerne hay
ad libitum (167 g CP and 502 g NDF per kg DM) with
free access to fresh water and mineral/vitamin blocks
were used. Rumen digesta was collected before
morning feeding, transferred into thermos flasks and
taken to laboratory to be filtered through cheesecloth
to obtain rumen fluid for the incubations.
In vitro incubations
The bioassay for the assessment of the activity of
tannins has been described in detail by Ammar et al.
(2004), using the in vitro gas production technique.
Samples of forages were weighted in serum bottles
that were sealed and incubated at 39 °C, either with or
without the addition of PEG (500 mg). The volume of
gas produced was recorded using a pressure transducer
and a calibrated syringe at 6, 12, 24 and 48 h after
inoculation. Gas production was corrected with the
values of the blank bottles (without substrate). The
increase in gas when PEG was added is a measure of
tannin activity (Makkar et al. 1995).
In vitro gas production technique was adapted from
that described by Theodorou et al. (1994) to estimate
fermentation kinetics. Samples (500 mg) were incu-
bated with 50 mL of buffered rumen fluid in serum
bottles. Blanks (bottles with only rumen fluid) were
included in each incubation batch. Bottles were closed
with rubber stoppers and sealed with aluminium caps
and placed in an incubator at 39 °C. The volume of gas
produced was recorded at 3, 6, 9, 12, 16, 21, 26, 31, 36,
Agroforest Syst
48, 72, 96, 120 and 144 h using a pressure transducer
and a calibrated syringe.
In vitro fermentation end-products were assessed in
24-h batch cultures. Samples (400 mg) of each
substrate were incubated in serum bottles filled with
40 mL of buffered rumen fluid at 39 °C. After 24 h of
incubation, the volume of gas produced was measured
as described above and a sample of gas was taken for
the analysis of methane. Bottles were opened, pH was
measured and a sample (0.8 mL) was added to a
deproteinising solution (0.5 mL) for volatile fatty
acids analysis. Methane in fermentation gas was
determined by gas chromatography (GC) using a
Shimadzu GC-14 B GC (Shimadzu, Japan) equipped
with CarboxenTM 1000, 45/60, 2 m 9 3.2 mm col-
umn (Supelco, USA) and flame ionization detector as
described by Garcia-González et al. (2008a). Volatile
fatty acids were analysed by gas chromatography
using crotonic acid as internal standard (Garcia-
González et al. 2008b).
In vitro DM digestibility was determined using the
ANKOM-DAISY procedure (Ammar et al. 1999). The
procedure followed general conditions described in
the standard in vitro digestibility method (Goering and
Van Soest 1970). Samples of each substrate (400 mg)
were weighted into fibre bags (#57 bags; ANKOM
Technology Corporation, Fairport, NY, USA). Bags
were sealed and incubated in buffered rumen fluid for
48 h at 39 °C. Bags were washed in cold water and
subsequently extracted with boiling neutral detergent
for 1 h to determine the undigested residue and to
estimate in vitro digestibility (Ammar et al. 1999).
Calculations and statistical analysis
Gas production data were fitted using the exponential
model proposed by France et al. (2000):
h i
G ¼ A 1eð c ðtLÞÞ for t  L
where G (mL/g) denotes the cumulative gas produc-
tion at time t; A (mL/g) is the asymptotic gas
production; c (h-1) is the fractional fermentation rate
and L (h) is the lag time. Extent of DM degradation in
the rumen (dg, %) was calculated as proposed by
France et al. (2000).
Data were subjected to analysis of variance per-
formed using the GLM procedure of SAS, with browse
species and PEG addition as fixed effects and with
source of inoculum as a blocking factor (random
effect). The Tukey test was used for the multiple
comparison of means.
Results and discussion
Chemical composition of the plant material collected
from the different species is shown in Table 1. The CP
content of the plant species varied widely, being
particularly high for deseeded pods of A. julibrissin
and leaves of V. faba and low for the pericarp of P.
granatum and barks of C. azel. The fibre contents
ranged from 179 to 513 g NDF/kg DM, and from 113
to 407 g ADF/kg DM. The highest value of ADL was
observed for bark of C. azel (223 g/kg DM). CP and
NDF concentrations influenced the extent of ruminal
fermentation of organic matter (OM) and thus the
amount of end-products (in particular VFA) released
(Njidda and Nasiru 2010).
Tannin composition of the plant species is shown in
Table 2. The highest concentrations of total
extractable phenols and tannins were observed in P.
granatum leaves and pericarp, whereas A. halimus
showed the lowest concentrations. A. nilotica, C. azel
and A. julibrissin showed the highest, and A. halimus
and A. herba-alba the lowest total condensed tannin
contents.
For screening plants for potential antimethanogenic
activity, a proposed approach is to perform a tannin
bioassay jointly with phenolic and tannin chemical
analyses (Ammar et al. 2004; Jayanegara et al. 2009).
Table 3 shows the effect of adding PEG to batch
cultures on fermentation gas production after 6, 12, 24
and 48 h of in vitro incubation of the plant samples in
buffered rumen fluid. The response to PEG treatment
(represented by the increase over the value without
PEG addition) varied with the incubation time. The
greatest response to PEG was recorded with C. azel
barks at 24 h and A. nilotica pods at 6 h of incubation.
The response to PEG treatment declined with incuba-
tion time, except for the pericarp of P. granatum and
C. azel. Screening plants for the biological activity of
tannins using a bioassay (gas production with and
without PEG) seemed to be a better alternative than
chemical analyses (Mlambo et al. 2009). Condensed
tannins limit in vitro fermentation, thus the increase in
gas production following inclusion of PEG provides a
measure of potential effects of tannins on nutrient
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Table 1 Chemical composition of the browse plants

Plant species Dry matter (g/ Dry matter (g/kg)
kg)
Organic Neutral detergent Acid detergent Acid detergent Crude
matter fibre fibre lignin protein

Albizia julibrissin 904 927 365 278 85 242

Punica granatum 919 956 277 169 50 34
(pericarp)
Punica granatum 915 911 222 155 95 109
(leaves)
Vicia faba 888 857 179 121 40 194
Atriplex halimus 871 815 253 113 47 157
Acacia nilotica 909 929 323 227 94 167
Artemisia herba-alba 901 920 359 273 115 123
Calligonum azel 916 891 513 407 223 57

Table 2 Concentrations of phenolic compounds (g standard equivalent*/kg DM) in browse species

Plant species Total Total Bound condensed Free condensed Total condensed
extractable phenols extractable tannins tannin tannin tannin

Albizia julibrissin 77 62 110 545 655

Punica granatum 286 281 37 188 224
(pericarp)
Punica granatum 312 306 68 11 79
(leaves)
Vicia faba 65 48 157 21 178
Atriplex halimus 26 22 46 3 49
Acacia nilotica 114 111 148 703 851
Artemisia herba-alba 41 37 47 25 72
Calligonum azel 146 137 148 570 719
*Total phenols and tannins were expressed as tannic acid equivalent and condensed tannins as leucocyanidin equivalent

degradability (Makkar 1988). The highest gas pro-
duction value (P \ 0.05) was observed in V. faba
(Table 3). This can be attributed to its lower con-
densed tannins and lignin but highest CP content
(Tables 1 and 2). Comparable results were obtained
with pods and leaves of acacia species (Alam et al.
2007; Mlambo et al. 2008; Bouazza et al. 2012).
Biological activity of tannins has been reported to vary
among forage species due to the chemical structure
and nature of tannins (Rubanza et al. 2005) and degree
of polymerization (Schofield et al. 2001). In vitro
studies show that some tannins are more active than
others (Aerts et al. 1999; Osborne and McNeill 2001;
Bueno et al. 2008). The PEG-based in vitro tannin
bioassay complements the chemical methods because
it specifically highlights the presence of potential
active tannins, whereas tannin concentration is
reported not to be always a good predictor of their
biological activity (Mlambo et al. 2009; Vitti et al.
2005). Condensed tannins build complexes with
different cell plant constituents and digestive enzymes
thus decreasing digestibility (Waghorn 2008). The
factor limiting rumen fermentation and digestion is the
interaction between tannins and feed constituents such
as structural (cellulose, hemicellulose, pectins) and
non-structural carbohydrates and all proteins
(McSweeney et al. 2001; Jayanegara et al.
2012, 2015), so that PEG binds tannins preventing

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Table 3 Gas production (mL/g dry matter incubated) of the browse plant species
Incubation Albizia Punica Punica Vicia Atriplex Acacia Artemisia Calligonum SEM P value
time (h) julibrissin granatum granatum faba halimus nilotica herba-alba azel
pericarp leaves Plant PEG Plant 9 PEG
species

6 w/o 17.2d 43.6b 30.2c 80.3a 9.4de 4.0ef 17.9d 0.0g 5.2 \ 0.001 \ 0.001 \ 0.001
PEG
w/ 37.6c 55.9b 34.7c 79.8a 8.1e 36.1c 20.8d 0.0f 4.9
PEG
12 w/o 56.6d 97.6b 75.7c 150a 50.1d 23.7e 54.8d 0.0f 8.9 \ 0.001 \ 0.001 \ 0.001
PEG
w/ 96.3c 110b 84.8c 149a 46.8d 89.7c 59.5d 13.0e 8.1
PEG
24 w/o 109c 136b 132b 192a 109c 63.9d 108c 1.5e 10.9 \ 0.001 \ 0.001 \ 0.001
PEG
w/ 145c 163b 138c 193a 108d 145c 114d 28.0e 8.6
PEG
48 w/o 131c 149bc 161b 208a 149bc 84.3d 134bc 8.9e 11.7 \ 0.001 \ 0.001 \ 0.001
PEG
w/ 173bc 197ba 171bc 225a 150cd 180bc 135d 44.6e 10.7
PEG
w/o PEG or w/PEG: incubated either without or with polyethylene glycol, respectively
SEM Standard error of the mean
a,b,c,d
means in the same row with different superscripts are significantly different (P \ 0.05)

123

its binding to feed constituents thus suppressing their
effects on digestion. The main interaction between
tannins and feed molecules is via hydrogen bonds
(Mueller-Harvey 2006).
Table 4 shows that the addition of PEG affects
(P \ 0.05) the asymptotic gas production (A) and
fractional rate of fermentation (c) to a lesser extent
than gas production measurements at certain incuba-
tion times. The lowest (P \ 0.05) A, c and dg values
were for C. azel in absence and presence of PEG,
showing that this is a rather undegradable material. In
contrast, Vicia faba foliage was the most degradable
substrate, with the highest A, c and dg values.
Guimarães-Beelen et al. (2006) suggested that when
the rate of gas production is reduced, the bacteria
proliferation is restricted. Elahi et al. (2014) explained
that tannin complexes limit the attachment of bacteria
to the feed components.
Despite the increase in gas production upon the
addition of PEG, in vitro digestibility was not
significantly affected (P \ 0.05) by the addition of
PEG (Table 4). Similar observations have been
reported elsewhere (Makkar et al. 1995; Getachew
et al. 2000b; Osuga et al. 2008; Bouazza et al. 2014).
This could be due mainly to the tannin-PEG com-
plexes, which become insoluble in neutral detergent
solution, thus distorting the weight of the incubation
residue (Osuga et al. 2008). However, the chemical
structure, concentration and biological effects of
tannins in forages, and their nutritional value, show
large variability (McSweeney et al. 2001).
Methane production was lowest when C. azel bark
or A. halimus foliage were fermented in vitro
(Table 5). C. azel bark is a low degradable substrate,
and less methane is produced just because this material
is fermented only to a minor extent. When compared
the values with or without the addition of PEG, it
seems that tannin has a depressing activity on methane
production when either A. julibrissin or A. nilotica are
fermented. Other acacia species seem to be effective
reducing methane in the rumen (Grainger et al. 2009).
The inhibitory effects of condensed tannins on
methanogenesis have been attributed to their direct
effects on rumen methanogenic archaea and protozoa
(Patra and Saxena 2009), indirectly leading to a
depression of fibre degradation (Tiemann et al. 2008;
Patra and Saxena 2011). Any reduction in fibre
degradation is likely to reduce methane by limiting
the availability of H2 as a substrate for methanogenesis
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Agroforest Syst
(Moss et al. 2000; Jayanegara et al. 2011). Tannins are
known to decrease protozoa (Bhatta et al. 2009) which
are in close association with methanogens (Morgavi
et al. 2010). The magnitude of decrease due to
condensed tannins in the present study was relatively
higher than in other studies using tannins extracted
and purified from plant leaves (Jayanegara et al. 2015).
Different forms of tannins (whole plants or extracted
tannins) may influence the CH4 emissions and rumen
fermentations parameters differently, most probably
affecting the magnitude of the effects (Jayanegara
et al. 2011). The reduced methane production when A.
halimus was incubated is in agreement with results
reported by other authors (Soltan et al. 2012; Medjekal
et al. 2018). In this particular case, the effect cannot be
attributed to phenolic compounds or tannins, as these
were very low in Atriplex. In the whole plant, rumen
methanogenesis can be affected not only by tannins,
but also by other components such as fibre (Beau-
chemin et al. 2008), lipids (Machmüller et al. 2000),
saponins (Hess et al. 2003) or essential oils (Benchaar
et al. 2008). Furthermore, Atriplex foliage was a
highly degradable material (based on gas production
kinetics and in vitro digestibility coefficients), sug-
gesting that the observed low methane production
could be due to a specific effect on methanogenesis
rather to a broad inhibitory effect on ruminal fermen-
tation. If this effect could be further confirmed, A.
halimus could be a promising highly digestible
roughage limiting the methane emissions by
ruminants.
Ruminal VFA concentrations mainly indicate the
degradation patterns of carbohydrates by microbes.
Based on total VFA, acetate and propionate concen-
trations, V. faba leaves seemed to be the most
fermentable substrate (Table 5). The highest acetate
to propionate ratio was observed with C. azel bark.
High acetate to propionate ratios will indicate a more
acetogenic fermentation, due to the activity of
fibrolytic bacteria degrading substrates rich in struc-
tural carbohydrates (Getachew et al. 2004). The
depressing effects of tannins on ruminal fermentation
of A. nilotica pods are revealed from the substantial
increase in total and individual VFA concentrations in
response to the addition of PEG. This finding was
consistent with other researchers using tannin-rich
species in their studies (Getachew et al. 2008; Singh
et al. 2012; Goel and Makkar 2012), in which total
VFA production was limited when tannin-rich tropical
 Agroforest Syst

Table 4 In vitro gas production kinetics and digestibility of the browse plant species
Parameter Albizia Punica Punica Vicia Atriplex Acacia Artemisia Calligonum SEM P value
julibrissin granatum granatum faba halimus nilotica herba-alba azel
pericarp leaves Plant PEG Plant 9 PEG
species

A (mL/g) w/o PEG 180bc 178bc 203cd 223d 194c 152b 178bc 66.9a 5.5 \ 0.001 0.318 0.823
bc bc cd d bc b bc
w/PEG 177 189 203 231 192 159 187 62.3a 6.7
c (h-1) w/o PEG 0.052c 0.067d 0.043c 0.097e 0.042c 0.028b 0.053c 0.013a 0.0026 \ 0.001 \ 0.001 0.100
d d cd e bc ab cd
w/PEG 0.050 0.052 0.043 0.086 0.035 0.024 0.046 0.012a 0.0023
L (h) w/o PEG 0.96ab 0.00a 0.00a 0.19a 3.30b 1.00ab 1.19ab 0.92ab 0.548 \ 0.001 0.534 0.904
a a a a a a a a
w/PEG 1.20 0.00 0.10 0.33 2.61 1.42 0.40 0.00 0.623a
dg (%) w/o PEG 44.1c 54.5d 52.4d 69.7e 45.7c 29.7b 47.4c 12.9a 0.85 \ 0.001 \ 0.001 0.247
w/PEG 42.2c 49.5de 52.0e 67.2f 42.6c 26.7b 45.2cd 12.8a 0.83
AIVD (%) w/o PEG 68.8ef 59.6de 53.3cd 72.0f 66.3ef 37.5b 45.6bc 27.1a 3.89 \ 0.001 0.106 0.410
d c c d d b b
w/PEG 70.7 59.2 59.0 72.5 69.1 39.8 48.0 24.0a 4.13
TIVD (%) w/o PEG 73.9d 74.4d 80.0e 87.4f 83.6ef 51.1b 60.4c 44.3f 3.80 \ 0.001 0.015 0.330
c c e e e b c
w/PEG 75.7 76.0 82.2 86.8 85.6 54.3 60.4 43.8a 3.84
A asymptotic gas production, c fractional rate of fermentation, L Lag time, dg rumen degradability, AIVD apparent in vitro digestibility, TIVD true in vitro digestibility
w/o PEG or w/PEG, incubated either without or with polyethylene glycol, respectively
SEM standard error of the mean
a,b,c,d,e,f
means in the same row with different superscripts are significantly different (P \ 0.05)

123
123
Table 5 Fermentation end-products (methane in mmol/g dry matter incubated and volatile fatty acid (VFA) concentrations in mmol/L) when browse plant species were
incubated in vitro in buffered rumen fluid for 24 h
Albizia Punica Punica Vicia Atriplex Acacia Artemisia Calligonum SEM P value
julibrissin granatum granatum faba halimus nilotica herba- azel Plant PEG Plant 9 PEG
pericarp leaves alba species

Methane w/o PEG 2.55b 2.63b 1.12cd 4.92a 0.93d 1.72c 1.63c 0.13e 0.094 \ 0.001 \ 0.001 \ 0.001
c d e a d b e
w/PEG 3.22 2.45 0.88 4.67 2.00 3.81 1.04 0.01f 0.080
Acetate w/o PEG 31.5b 23.0de 21.4e 38.5a 26.9c 21.2e 24.4d 13.6f 0.31 \ 0.001 \ 0.001 \ 0.001
b d f a cd c e
w/PEG 32.3 27.1 19.6 38.6 27.6 28.5 22.2 16.7g 0.14
Propionate w/o PEG 11.8a 6.93b 6.12c 12.2a 7.35b 7.49b 6.24c 3.35d 0.092 \ 0.001 \ 0.001 \ 0.001
a c e a d b e
w/PEG 12.2 8.37 5.17 12.1 7.56 9.45 5.39 3.97f 0.095
Isobutyrate w/o PEG 0.75ab 0.38c 0.46c 0.94a 0.57bc 0.54cd 0.75ab 0.49c 0.028 \ 0.001 \ 0.001 0.002
w/PEG 0.88a 0.57c 0.49c 0.89a 0.62bc 0.80ab 0.76ab 0.55c 0.026
Butyrate w/o PEG 2.64d 3.66b 2.83d 4.69a 3.23c 1.69e 2.69d 1.67e 0.045 \ 0.001 \ 0.001 \ 0.001
d b e a d c f
w/PEG 3.34 4.33 2.80 4.67 3.38 3.67 2.51 2.20g 0.021
Isovalerate w/o PEG 0.94c 0.52e 0.68d 1.30a 0.93c 0.76d 1.18b 0.68d 0.018 \ 0.001 \ 0.001 \ 0.001
ab d d a c ab b
w/PEG 1.23 0.68 0.73 1.34 0.88 1.22 1.14 0.99c 0.021
Valerate w/o PEG 0.54b 0.33d 0.36cd 1.36a 0.53b 0.47bcd 0.50bc 0.33d 0.021 \ 0.001 \ 0.001 \ 0.001
bc d d a cd b cd d
w/PEG 0.71 0.43 0.39 1.39 0.49 0.78 0.50 0.38 0.034
Total VFA w/o PEG 48.2b 34.8d 31.8e 58.9a 39.5c 32.2e 35.8d 20.2f 0.25 \ 0.001 \ 0.001 \ 0.001
w/PEG 50.6b 41.4d 29.2f 59.0a 40.5d 44.4c 32.5e 24.8g 0.15
Acetate to w/o PEG 2.68d 3.32bc 3.50ab 3.16bcd 3.66ab 2.84cd 3.92a 4.08a 0.085 \ 0.001 0.020 0.158
propionate e d bc d c d ab
w/PEG 2.64 3.24 3.78 3.18 3.65 3.02 4.12 4.20a 0.049
ratio
w/o PEG or w/PEG, incubated either without or with polyethylene glycol, respectively
SEM standard error of the mean
a,b,c,d
means in the same row with different superscripts are significantly different (P \ 0.05)
Agroforest Syst
Agroforest Syst
species are fermented. For most of the forages tested,
the results are in agreement with studies in sheep
(Priolo et al. 2000) and goats (Silanikove et al. 2006)
where VFA concentrations were not affected when
animals received the same diets but supplemented
with PEG concentrations. Effects on VFA concentra-
tion could be also due to the presence of other
secondary metabolites that can affect ruminal fermen-
tation negatively (Rira et al. 2015).
Conclusions
In conclusion, all the browse species used in the
current study, with the exception of Calligonum azel
bark, can be used as alternative feedstuffs for ruminant
nutrition. Vicia faba was the most digestible forage.
Albizia julibrissin and Acacia nilotica are also
digestible roughages rich in protein, although their
use in ruminant diets can be restrained by their high
tannin contents. Methane production with Calligonum
azel bark is very low, but because this is a rather
indigestible material. The most promising forage in
terms of reduced methane emissions is Atriplex
halimus forage, because the decreased methane pro-
duction is not associated to a reduced rumen degra-
dation and fermentation of this forage in the rumen.
Compliance with Ethical Standards
Human and animal rights Animals were handled and cared
in accordance with the Spanish guidelines for experimental
animal protection (Royal Decree 1201/2005) and experimental
protocols were approved by the Institutional Animal Care and
Use Committee of the University of León.
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